Dissertations / Theses on the topic 'Lipooligosaccharide'

Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of Haemophilus and Neisseria. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of H. somnus, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to H. somnus LOS were produced and used with cross-reacting MAbs to H. aegyptius LOS (MAb 5F5) and Neisseria gonorrhoeae LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of H. somnus. Using three MAbs, thirty-nine of these H. somnus strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of H. somnus strains. The anti-H. somnus LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the H. somnus strains. Six strains (13%) were not recognized by any of these MAbs.

Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in H. somnus LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed.

Master of Science

Targeted delivery of drugs directly to the lung epithelium is a promising, though challenging, strategy for the treatment of diseases that affect the lung tissues, such as infections caused by cell-penetrating pathogens, cystic fibrosis, and cancer. With appropriate surface functionality, such as through the attachment of ligands that recognize receptors on cellular surfaces, particulate carriers show improved efficiency in penetrating cells in vitro. A useful class of ligands is produced by many natural human pathogens that infect the respiratory tract. A variety of phylogenetically distinct respiratory bacterial pathogens, such as Haemophilus influenzae, invade host cells in the upper airways by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms. By expressing host carbohydrate structures, including phosphorylcholine (ChoP), as a terminal structure on the LOS, the bacteria exhibit molecular mimicry of the host and are able to evade the host immune system. The effectiveness of LOS to induce cellular uptake of the bacteria is dependent on the specific glycoform, with higher ChoP content inducing more bacterial adherance into the lung epithelial. These ligands naturally expressed on bacterial cell surfaces can be isolated and utilized as targeting ligands for delivery vehicles. The studies described in this thesis focus on the development of particulate drug carriers coated with LOS bacterial ligands to enhance the targeting and binding of the carriers to the lung epithelium. Three NTHi clinical isolates were screened to select the strain with the highest ChoP level, and NTHi 3198, an isolate from a patient with chronic obstructive pulmonary disease (COPD), was selected due to its high ChoP activity. LOS from NTHi 3198 was isolated from the bacterial cell membrane, and its activity verified using dot immunoblot and ELISA techniques. Particles (0.2 and 1 µm) composed of polystyrene or poly(lactic-co-glycolic acid) were passively coated with 0.005-50 µg/mL of the isolated LOS 3198 with or without gelatin, coated with gelatin alone, or left uncoated. The LOS coating on the particles was verified using either XPS or ELISA. The association of particles with human bronchial epithelial cells was investigated using two cell culture models, 16HBE14o- and Calu-3, as a function of particle concentration and incubation time. The expression of PAFR on both cells types was confirmed, though the expression of PAFR on 16HBE14o- cells was significantly greater than on Calu-3 cells. Enhancement of 0.2 µm particle-cell association was achieved through coating of the particles with LOS. However, no significant difference in particle-cell association was observed for the 1 µm particles based on particle coating. Control particles of 0.2 µm size, those coated with gelatin (with or without LOS) or uncoated, exhibited low cell binding with a maximum of about 10-18% of cells associated with particles. The ability of the LOS ligand to enhance particle-cell association was coating concentration dependent, with a low coating concentration of LOS having little effect on association, but a concentration 1000-fold higher causing a doubling of the percentage of cells associated with particles at 24 hours. This enhancement was attributed to increased cellular binding of the 0.2 µm particles to the cell surface by confocal microscopy, and was further increased by activating the PAFR prior to incubation with particles. These results suggest the potential application of LOS as a targeting ligand for lung epithelial cells, especially under conditions where PAFR has been activated, such as occurs in lungs infected with Haemophilus influenzae. A significant reduction in particle-cell association was observed when particles were incubated with Calu-3 cells due to the presence of mucus on the cellular surface. This suggests that further optimization of the drug carrier system is needed to efficiently overcome the mucosal fluids.

Thesis (Ph. D.)--Illinois State University, 2005.
Title from title page screen, viewed September 27, 2006. Dissertation Committee: Wade Nichols (chair), Jon Friesen, Craig Gatto, Laura Vogel, Brian Wilkinson. Includes bibliographical references (leaves 87-93) and abstract. Also available in print.

Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

The enteric pathogen, Campylobacter jejuni, produces a range of LOS structures, however, the precise functions of LOS molecules in infection are largely undetermined. LOS structural diversity is known to arise from variation in LOS biosynthesis gene content and gene sequence. In determining the extent of LOS biosynthesis gene content variation in a group of mainly clinical C. jejuni isolates, in this study two new clusters of LOS biosynthesis genes have been identified. The C. jejuni LOS core can also undergo phase variation due to the presence of GC homopolymeric tracts in the protein coding sequence of biosynthesis genes in the cluster. Therefore, the variation in homopolymeric tract length was investigated in five genes including those in the LOS biosynthesis cluster. Many bacteria are known to vary LPS or LOS structure in response to different environment stimuli. Following the identification of a number of promoters in the LOS core biosynthesis cluster, promoter activity was measured following growth under several different conditions. Although promoter expression did not vary extensively with the different environmental stimuli, less LOS was extracted from cell grown under iron-limitation. The other aim of this work was to investigate LOS biosynthesis gene function. The genes waaF and lpxL are involved in core and lipid A biosynthesis respectively. Mutation of both genes had a substantial effect on LOS core structure and preliminary studies indicate both genes are important for adhesion and invasion of human intestinal cells. The deletion of several genes from the general protein glycosylation pathway also led to the truncation of the LOS core, indicating overlap between the two carbohydrate biosynthesis pathways.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Campylobacter jejuni is an enteric bacterium that causes human gastroenteritis worldwide. Some C. jejuni strains exhibiting human ganglioside-like lipooligosaccharide (LOS) structures, such as GM1 ganglioside, can induce an autoimmune neuropathy of the peripheral nervous system known as the Guillain-Barré syndrome (GBS). This GBS-inducible determinant is encoded by a gene cluster, which shows a high degree of variation among C. jejuni strains. The experiments presented in this thesis were conducted to give a better insight into the LOS synthesis genes in relation to the pathophysiology of C. jejuni. Firstly, a C. jejuni strain without GM1-like molecules was shown to be able to take up large DNA fragments, including LOS synthesis genes, from a strain expressing GM1-like molecules and consequently be transformed into a number of potential GBS-inducible transformants, which exhibited a high degree of genetic and phenotypic diversity. The ability of C. jejuni to take up and integrate foreign DNA explains the genome plasticity observed in this pathogen. Secondly, while attempting to analyse transcription of the LOS gene cluster, neither published methods nor any commercially available kits for RNA isolation could produce DNA-free RNA from C. jejuni. Combinations of these methods were trialled and only the combination of RNAzolB, TURBO DNase treatment, and acid phenol extraction was able to produce DNA-free RNA. The RNA isolated from most C. jejuni strains showed different RNA patterns to that of other bacteria. In addition the RNA from C. jejuni seemed closely associated with DNA compaired to RNA from other organisms. This might be caused by species-specific DNA conformation or chromatin structure. Thirdly, bidirectional transcription was observed in the LOS gene cluster. Both DNA strands were transcribed but transcription of the non-coding strands was at a lower rate, and both sense and antisense transcripts of each LOS gene tested were responsive to acid stress. This unusual transcription might have a potential effect on the expression of the GBS-inducing determinant. Finally, one of the LOS genes, the htrB gene, was further analysed. It was shown that expression of the htrB gene affects morphology, viability, growth ability, and sensitivity to stress environments. These results showed that the LOS molecule of C. jejuni is involved in many processes and is an important molecule for survival.

Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis world wide and has been linked to several severe complications including autoimmune syndromes which can result in paralysis. Despite being the subject of much study, C. jejuni remains a major public health burden in both developing and developed nations. There is currently no vaccine available for protection against this pathogen and the mechanisms important for C. jejuni pathogenesis are not fully defined. This study has employed a range of experimental approaches to investigate the molecular mechanisms involved in C. jejuni pathogenesis. Lipooligosaccharides (LOSs) are surface structures and known virulence factors of C. jejuni which are involved in serum resistance, resistance to phagocytic killing, endotoxicity and adhesion. Mutagenesis studies targeting the putative LOS biosynthesis genes wlaRF, wlaTA, wlaTB, wlaTC and waaV were performed in order to characterise the proteins encoded by each of these six genes and assess their potential role in C. jejuni pathogenesis in vitro. The gene product of wlaTA was found to be essential for C. jejuni survival and therefore a knock out mutant could not be generated. Phenotypic characterisation of four knock-out mutants confirmed that each gene contributed to the construction of the LOS molecule as all four mutants produced a truncated LOS moiety and altered their immunoreactivity. Further analysis determined that the production of complete LOSs was important for C. jejuni to invade and adhere to both human and chicken cells in vitro. This study identified a link between the inactivation of two LOS biosynthesis genes and the loss of motility, another important virulence factor. A major source of human C. jejuni infection is contact with contaminated poultry. However, C. jejuni exists as a commensal in chickens. It is currently not known why C. jejuni is pathogenic to humans and not to chickens and the differences between these two hosts represent pathogenic and non-pathogenic environments respectively. These environmental differences were exploited in this study. The four conditions investigated were temperature, blood, bile and host cells in vitro. Five different C. jejuni strains (NCTC11168, 81116, HB93-13, a recent human enteritis isolate and a recent chicken isolate) were subjected to modelled

Glycosyltransferases are a diverse group of enzymes found in natire and perform critical functions in all domains of life by transferring sugars to various targets, forming glycoside bonds. In Gram-negative bacteria such as Moraxella catarrhalis, glycosyltransferases are essential in the biosynthesis of the lipooligosaccharide (LOS) structure, a major component of the outer membrane. M. catarrhalis is a frequent coloniser of the human upper respiratory tract and is associated with ear infections and diseases of the lower respiratory tract, particularly in patients with underlying lung impairment. M. catarrhalis strains omly display three major LOS serotypes: A, B and C. The genetic basis of M. catarrhalis LOS biosynthesis has been elucidated and functions suggested for each of the identified LOS glycosyltransferases.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
Full Text

Repetitive tetramers of the DNA sequence 5'-CAAT-3' are present in several loci associated with lipooligosaccharide (LOS) phase variation in Haemophilus influenzae type b (Hib). In an attempt to identify H. somnus phase-variable LOS genes, the presence of CAAT repeats within the H. somnus 738 genome was confirmed using a (CAAT)7 probe. A 3.9 kb EcoRI fragment that reacted with the probe was cloned and sequenced. Sequence analysis confirmed the presence of 31 CAAT repeats downstream of two potential start codons, and indicated that small or large proteins would be encoded depending on the number of CAAT repeats. The larger gene products showed 46% amino acid homology to Lex2b from Hib, which influences LOS phase variation in that species. In H. somnus, this gene was named lob1 (lipooligosaccharide biosynthesis gene). Sequence analysis showed that randomly selected colonies most frequently contained 33 CAAT repeats in lob1, corresponding to a 294 amino acid product. Colonies selected for negative reactivity to mAb 5F5 were significantly more likely to have different numbers of CAAT repeats in lob1 than randomly selected colonies. The presence of lob1 in trans altered the LOS profile of a non-phase variable strain of H. somnus, and caused increased levels of reactivity to polyclonal antisera made to purified LOS from strain 738. Based on the ability of this gene to alter the LOS profile of a non-phase varying strain and the correlation of changes in CAAT repeats with mAb 5F5 reactivity, lob1 appears to be involved in LOS biosynthesis and phase variation.
Master of Science

Lipooligosaccharides (LOS) are an important constituent of the bacterial outer membrane involved in maintaining cellular structural integrity. In Campylobacter jejuni LOS is postulated to have a role in the virulence of this important gastrointestinal pathogen. The ability of C. jejuni to vary LOS structure and the molecular mimicry of host gangliosides by LOS epitopes suggests a role in the avoidance of host defence mechanisms. Molecular mimicry may form the immunopathological basis for an association with Guillain Barre syndrome. LOS structures differ between strains due to gene content, sequence and tract variation in the LOS biosynthesis gene cluster. Studies have examined the extent of gene content variation in the LOS gene cluster and showed gene content in this region can vary extensively. The aim of this work was to search for novel gene content and further investigate the role of LOS in C. jejuni. Both PCR and microarray-based methods were developed to classify 50 clinical strains by LOS gene content. Several strains had gene content that did not appear to correlate with any known class and further detailed analysis highlighted that variation is grater than previously supposed. Sequencing revealed two strains that contained capsule polysaccharide biosynthesis genes within the LOS biosynthesis cluster. The presence of capsule genes within the LOS biosynthesis cluster highlights the potential for recombination between these variable polysaccharide loci and further extends the possibility of interaction between the capsule and LOS polysaccharides.;A LOS large deletion mutant was constructed in the genome strain, NCTC 11168 removing the genes between wlaA and wlaT. This mutant shared characteristics of other deep rough mutants being slow growing and sensitive to antibiotics and detergents. Construction of this mutant confirmed the minimal LOS core biosynthesis gene content for bacterial viability and showed the importance of LOS for this bacterium through a role for LOS in host cell invasion.

Virulent strains of the bovine opportunistic pathogen Haemophilus somnus (Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. It is reported in this dissertation that H. somnus significantly (P <0.001) inhibited production of superoxide anion (O2-) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O2- production was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide (LOS), or cell-free supernatant from mid-log phase cultures did not inhibit O2- production, indicating that O2- inhibition required contact with live H. somnus. Commensal isolates of H. somnus were less capable or incapable of inhibiting macrophage O2- production compared to isolates tested from disease sites. H. somnus shares conserved epitopes in its LOS with Neisseria gonorrhoeae, N. meningitidis, and H. influenzae, and can also undergo structural phase variation of these LOS epitopes. Sialylation of the terminal galactose of H. somnus LOS is another reported virulence mechanism. Current sequencing of the genomes of H. somnus strains 2336 (pathogenic) and 129Pt (commensal) has enabled in silico identification of three open reading frames (ORFs) involved in sialylation. The ORFs-1 (hsst-I) and -2 (hsst-II) had BLASTx homology to sialyltransferases, while ORF-3 (neuAhs) had BLASTx homology to CMP-sialic acid synthetases. These ORFs were amplified by PCR and cloned into the expression vector pCWOri+. Thin layer chromatography of the hsst-I gene product showed this sialyltransferase exhibited preference for sialylation of terminal N-acetyllactosamine (LacNAc, beta-Gal-[1,4]-beta-GlcNAc-R). However, Hsst-II preferentially sialylated lacto-N-biose (LNB, beta-Gal-[1,3]-beta-GlcNAc-R). In this study, phase variation of the terminal linkage in isolate 738 from a 3 linked galactose (LNB) to a 4 linked galactose (LacNac) was demonstrated. Such variation of a glycose linkage appears to be a novel mechanism of LOS phase variation. Furthermore, the ability of sialylated strain 738 LOS vs de-sialylated strain 738 LOS to induce Toll-like receptor 4 signaling was decreased by 28%, as determined by ELISA for Macrophage Inflammatory Protein-2. Therefore, sialylated LOS may aid H. somnus to avoid host innate immunity.
Ph. D.

Incorporation of N-acetyl neuraminic acid (NANA), or sialic acid, onto lipooligosaccharide (LOS) enhances the virulence of several bacterial species. In the present study, we assessed the effect of sialylation of Histophilus somni LOS on complement-mediated killing, binding of complement factor H (which converts C3b to inactive C3b (iC3b) and inhibit the alternative complement pathway) to the bacteria, complement activation by the LOS, and phagocytosis and killing of the bacteria by bovine polymorphonuclear leukocytes (PMN). Killing of H. somni by alternative complement pathway was measured by incubation of sialylated or non-sialylated H. somni with antibody-free precolostral calf serum (PCS) followed by viable plate count. A complement dose-dependent response to killing of non-sialylated H. somni by PCS was observed. However, sialylated H. somni were significantly (P = 0.001) more resistant to killing at any of the concentrations of PCS used.

Sialylated H. somni LOS activated (P = 0.025) and consumed (P = 0.001) less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized sheep red blood cells or rabbit red blood cells, and by western blotting of C3 activation products. Sialylated H. somni bound more factor H than non-sialylated bacteria (determined by enzyme-linked immunosorbent assay) (P = 0.004), supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni inhibited both PMN phagocytosis of 3H-thymidine-labelled bacteria (P = 0.004) and intracellular killing of the bacteria (P = 0.0001), compared to non-sialylated bacteria. Therefore, sialylation of the LOS results in enhanced binding of complement factor H to the bacteria, resulting in diminished complement activation, resistance to complement-mediated lysis, and PMN phagocytosis and killing.

Master of Science

Campylobacterjejuni, a Gram-negative enteric pathogen, is the leading cause of bacterial gastroenteritis in the developed world. A C. jejuni strain 8 1-176 transposon library was used to screen for mutants over-producing a calcofluor white (CFW)-reactive polymer implicated in biofilm formation. This identified two lipooligosaccharide (LOS) core mutants: one defective for a two-domain glycosyltransferase (lgtF), and the other defective in a heptosyltransferase (waaF). To determine if other LOS core mutants displayed a similar phenotype, and to explore other biological outcomes of step-wise LOS truncations on C. jejuni stress resistance and pathogenesis, mutant strains defective for GaiT and CstII were also constructed. Silver stain and mass spectrometry analyses confirmed the sequential truncation of sialic acid (ΔcstII), galactose (ΔgalT), two glucoses (ΔlgtF), and heptose II (ΔwaaF). While the ΔlgtF and ΔwaaF mutants exhibited enhanced biofilm formation and ΔlgtF displayed increased sensitivity to complement killing, no effect for these phenotypes and only modest alterations in CFW reactivity were seen with partial outer core truncations. Deletion of LgtF had no effect on mouse colonization in vivo, or on invasion and intracellular survival in epithelial cells in vitro. In contrast, the ΔwaaF mutant exhibited a significant defect in intracellular survival in vitro. Interestingly, the mutants exhibited stepwise increases in susceptibility to the antimicrobial peptide LL-37, with /waaF and ΔlgtF being more susceptible and ΔgalT and ΔstII being more resistant than wild type. In contrast, all of the mutants were highly susceptible to polymyxin B. This is the first report of C. jejuni susceptibility to LL-37 and of LOS affecting polymyxin B resistance. Each of these appears to be independent of overt effects on outer membrane protein expression, membrane stability, or surface hydrophobicity. Together, our data indicate that the length and specific moieties of the LOS play important roles in C. jejuni biology, and suggest a dynamic interplay of the LOS with other stress resistance factors.

Histophilus somni virulence factors include expression and antigenic variation of lipooligosaccharide (LOS). Phosphorylcholine (ChoP) is often expressed on H. somni LOS and also undergoes antigenic variation. In this study, five genes that play a role in expression and antigenic variation of ChoP, lic1ABCD and glpQ, were identified in the genome sequence of H. somni through sequence homology with Haemophilus influenzae genes. The open reading frame (ORF) of lic1A contained a variable number of tandem repeats of the tetranucleotide unit 5'-AACC-3'. Slipped strand mispairing in the repeat region during replication leads to shifting the downstream reading frame in and out of frame with the start codon, thus controlling phase variation of lic1A expression. Removal of the repeats from lic1A, cloning the gene in E. coli, and performing a functional assay on the product indicated that lic1A encodes a choline kinase and that the repeats were not required for expression of a functional gene product. Variation in the number of repeats in lic1A correlated with the antigenic variation of ChoP expression in strain 124P, but not in strain 738. This result supported previous findings that antigenic variation of ChoP expression in strain 738 is controlled through extension/truncation of the LOS outer core. Therefore, these results indicated that the lic1ABCD and glpQ genes control expression and antigenic variation of ChoP on the LOS of H. somni and that there are two possible mechanisms for ChoP antigenic variation. The role of H. somni expression of ChoP in colonization of the host respiratory tract was also examined. Experimental infection in the natural host showed that the population of H. somni that expresses ChoP was enriched in the bacteria that colonized the respiratory tract. In addition, bacteria expressing ChoP were able to aggregate bovine platelets through binding to the platelet activating factor receptor (PAF-R), which is also present on epithelial and endothelial cells. These results indicated that ChoP may play a role in the process of colonization and subsequent systemic invasion of host tissues, which may occur through binding of ChoP to PAF-R. Bacteria that did not express ChoP were more prevalent in systemic sites, indicating that ChoP expression may be disadvantageous for the organism during systemic dissemination.
Ph. D.

Moraxella bovoculi is a Gram-negative microorganism that has shown potential to aide in the pathogenesis of infectious bovine keratoconjunctivitis (IBK) or ‘pink-eye’ in cattle. An ocular disease, IBK has shown to cause a significant economic loss to the cattle and dairy industry with reported losses of AUD 21 million annually to the beef industry in Australia.1 Infected animals show symptoms of keratitis, conjunctivitis, corneal ulceration and in severe cases, IBK can lead to permanent blindness.2 Currently, the only known cause for IBK is the Gram-negative bacterium Moraxella bovis. However, M. bovoculi is a recently isolated bacterium from calves which could also play a role in the pathogenesis of IBK with reports suggesting it may play a role in host colonisation.2-4 Currently, no investigations on the lipooligosaccharide (LOS) structure of M. bovoculi have been completed which is a major virulence factor found in Gram-negative bacteria.3,5,6 This following study aimed to further understand the biological role of LOS in M. bovoculi as well as compare it to the previously studied Moraxella species, M. bovis and M. catarrhalis. The first aim investigated the growth profile and biological activity of Moraxella bovoculi. The second aim investigated the extraction and purification of the LOS of M. bovoculi leading to the eventual analysis of the oligosaccharide structure using NMR spectroscopy. An investigation into the biological role of LOS in M. bovoculi examined the growth and biological activity. The growth characteristics of M. bovoculi showed that the bacterium reached its stationary phase at 15 hours. This information was exploited to optimise the yield of viable bacteria for future isolation and purification of LOS. Endotoxin activity was quantified using a Limulus Amebocyte Lysate (LAL) assay. The endotoxin activity of M. bovoculi is identical to M. bovis Epp63 Lgt2Δ strains but smaller than Mb25, L183/2 and Epp63 M. bovis strains as well as the wild-type M. catarrhalis O35E strain. An Auto-aggregation assay that measured sedimentation rates in liquid media were found to be similar to M. catarrhalis 035E strain. The percentage of adherent bacterial cells to HeLa and Chang cells was examined and revealed significantly decreased percentages towards all strains except the mutant M. bovis Epp63 Lgt2Δ and M. catarrhalis O35EkdtA strains. Susceptibility to the hydrophobic agents Tween 20 and Triton X-100 was analysed using a disk diffusion assay at a concentration of 5%. M. bovoculi is less sensitive to Tween-20 when compared to all M. bovis strains but is more sensitive when compared to M. catarrhalis O35E. M. bovoculi is less sensitive to Triton X-100 when compared to M. bovis Mb25, and M. catarrhalis O35EkdtA strains but is more sensitive when compared to M. bovis L183/2, Epp63Lgt2Δ and M. catarrhalis O35E. Susceptibility to a selection of nine antibiotic agents was also examined using a disk diffusion assay. M. bovoculi saw a significantly higher susceptibility to chloramphenicol when compared to M. bovis Epp63 strain. Conversely, M. bovoculi saw a significantly lower susceptibility to nalidixic acid when compared to M. bovis L183/2 strain. M. bovoculi presented a significantly higher susceptibility to novobiocin and polymyxin B when compared to M. bovis Mb25, L183/2, Epp63 and the mutant Epp63 Lgt2Δ strains. However, M. bovoculi saw a significantly lower susceptibility to rifampicin when compared to M. bovis Mb25 and Epp63 Lgt2Δ strains. Lastly, M. bovoculi saw a significantly higher susceptibility to vancomycin when compared to M. bovis Mb25, Epp63 and Epp63 Lgt2Δ but is not when compared to M. bovis L183/2. It was also analysed here that β-lactamase activity was observed in M. bovoculi when comparing Ampicillin and Penicillin G to the β-lactamase inhibitor, Amoxicillin/Clavulanic Acid presenting with a higher susceptibility. Lastly, a bactericidal assay using bovine serum was conducted using bacterial cell counts. No difference is observed between the concentrations 0% and 25% for M. bovoculi. The LOS of M. bovoculi was extracted using three protocols sequentially to identify which protocol would acquire the highest and purest yield to use for NMR analysis. These protocols included phenol/chloroform/petroleum ether method, the phenol/EDTA/TEA method and the hot phenol/water method in that order. It was determined that the hot phenol/water method gave the highest yield of LOS material once it was liberated of nucleic acid and protein contamination using DNase, RNase and Proteinase K. An SDS-PAGE confirmed that M. bovoculi was devoid of an O-antigen due to absence of higher molecular weight banding patterns confirming it to be a LOS structure. LOS was subjected to mild acid hydrolysis to cleave the Lipid A from the Oligosaccharide. The oligosaccharide was then purified further using size exclusion gel chromatography that determined the oligosaccharide to be high in molecular weight as it travelled down the Bio-gel P4 rapidly. The pure oligosaccharide sample was examined using 1H NMR spectroscopy for purity. Pure oligosaccharide samples were analysed using an Avance Bruker 600 MHz spectrophotometer using 1D and 2D NMR experiments. Spectral assignment of M. bovoculi determined that the oligosaccharide structure contains six sugar residues in addition to the Kdo residue. A central trifunctional α-glucopyranose residue is determined to be linked to the O-5 of the Kdo. The α-glucopyranose is linked to two β-glucopyranose residues at the four and six position carbons. The α-glucopyranose is also linked to a β-galactopyranose at the three-position carbon which extends to a α-galactopyranose from its six-position carbon. The α-galactopyranose extends further to another α-galactopyranose residue at the four-position carbon. The oligosaccharide of M. bovoculi had unusual features such as it is devoid of heptose residues as previously seen in species like M. bovis and M. catarrhalis. However, the structure did not contain any N-Acetylglucosamine residues unlike serotypes A and C of M. catarrhalis, and N-Acetylgalactosamine resides unlike M. bovis Epp63 and Mb25 strains.7,8
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
Full Text

This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of Neisseria gonorrhoeae, N. meningitidis and Streptococcus pyogenes on host epithelial cells and in vivo.

N. gonorrhoeae cause the sexually transmitted disease gonorrhoea and N. meningitidis is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, N. meningitidis generally induced an earlier response compared to N. gonorrhoeae, probably as a result of the meningococcal capsule. The role of N. meningitidis serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure.

S. pyogenes may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. S. pyogenes ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of S. pyogenes is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of S. pyogenes.

The evolution of ON/OFF switching phase variable loci is presumed to have arisen due to the need for bacterial populations to cope with uncertain environments where selection fluctuates between opposing pressures. One such selection is believed to be the presence of bacteriophage. Bacteriophage can influence bacterial evolution by forcing the development of bacteriophage resistance mechanisms within bacterial populations. However, resistance mechanisms can often come at a cost, such as reduced survival against the immune responses. As such, phase variation may circumnavigate this cost by generating heterogeneous populations containing both resistant and sensitive phenotypes. This study aimed to investigate the two known phase variable bacteriophage resistance genes in Haemophilus influenzae hsdM and lic2A, which encodes the methyltransferase of a type I restriction modification system and a lipooligosacharide biosynthesis associated glycosyltransferase respectively. Analysis of the diversity of repeat tract lengths and ON/OFF states of these genes across H. influenza genomes within GenBank revealed that while lic2A was ON in the majority of strains, hsdM was OFF. Thus lic2A may be consistently beneficial while the hsdM benefit is transient. Analysis of samples from patients with COPD, showed that lic2A was ON in the majority of samples, while hsdM may also be ON state in a number of samples. Although a resistance phenotype could be observed for lic2A, no resistance could be observed for hsdM. Therefore, the dynamics of bacteriophage spread through populations heterogeneous for lic2A was investigated. The heterogeneous populations generated by phase variation reduced bacteriophage dispersal through bacterial populations. This mechanism may also allow bacterial populations to adjust their heterogeneity levels to control bacteriophage densities. The heterogeneity may further create diverse bacteriophage densities across the bacterial macropopulation through resistant populations acting as a barrier. The results demonstrate the potential for phase variation to aid in the survival of bacterial populations against bacteriophage predation.

This project provides a novel insight into the genetics of complex surface structures present in the ST25 lineage of the multi-drug resistant bacteria, Acinetobacter baumannii. The thesis employs a newly developed tool, Kaptive, to aid global tracking efforts of this pathogen and uncovers new structure variants which inform future therapeutic options.

Moraxella bovis, Moraxella catarrhalis and Nontypeable Haemophilus influenzae (NTHi) are Gram-negative, oxidase positive, pathogenic microorganisms. M. bovis causes Infectious Bovine Keratoconjunctivitis, known (IBK) as ‘pink-eye’ in cattle worldwide. The disease is of economic importance as it leads to substantial economic loss in the cattle and dairy industries. There are antibiotic treatments available to treat M. bovis associated infections, but they tend to be ineffective at controlling disease outbreaks. Importantly, the current series of antibiotics used to treat IBK have shown occurrence of resistance due to beta-lactamase enzyme produced by the bacteria. There does exist a pilin-based vaccine for IBK which has been approved for use in Australia, although it is problematic because it is not protective against all strains of M. bovis. M. catarrhalis and NTHi are human respiratory tract opportunistic pathogens responsible for otitis media in children and exacerbate chronic obstructive pulmonary disease in adults. Similar to M. bovis, both bacteria have been shown to produce β-lactamase, which has led to the emergence of antibiotic resistance. There is no licenced vaccine for M. catarrhalis or NTHi infections. In the past two-decades studies on M. catarrhalis lipooligosaccharide (LOS) have suggested that this cell surface glycolipid could potentially be incorporated into vaccines. This is based on immunogenicity in a mouse model and role in adherence and invasion of host epithelia and serum resistance. It is also conserved among strains. M. bovis lipooligosaccharide (LOS) is not well studied. Structural analysis of wild-type M. bovis strain Epp63 oligosaccharide (OS from LOS) have identified the core OS as containing eleven sugar residues, including Kdo (the number of Kdo residues is still unknown) with a branched structure. Interestingly, this core OS has an unusual terminal open chain (1S)-GalaNAc residue and lacks heptose residues in its inner core. Recent studies have also elucidated the structure of the cell surface glycans in other strains (M. bovis Mb25 capsule and M. bovis Epp63 LOS), but whether the unusual structural features are present in other strains of M. bovis, was not known. This study elucidated the OS structure and identified the presence of capsular polysaccharide in M. bovis strains Mb25 and L183/2. NMR spectroscopy of the OS from M. bovis Mb25 and L183/2 showed that the structural characteristics of Epp63 and Mb25 strains are shared and that L183/2 OS lacks the terminal (1S)-GalaNAc residue. Strain L183/2 has the same capsular polysaccharide as strain Mb25, namely unsulfated chondroitin, whereas strain Epp63 does not express a capsule. The biological activity of M. bovis Epp63 LOS and the role LOS play in causing disease is not known, therefore a series of biological assays were investigated using OS mutants with varying glycan length. It was identified that LOS truncation affected M. bovis Epp63 susceptibility towards the antibiotics novobiocin and chloramphenicol, but not to nalidixic acid, polymyxin B, rifampin, Tween 20, Triton X-100 or vancomycin. Highly truncated Epp63 OS mutant was found to be susceptible to the bactericidal activity of bovine serum. The growth rate of wildtype L183/2 was significantly slower than that of wild-type Mb25 or Epp63, however, the Epp63 OS mutants showed reduced growth rates compared to the wild-type. Limulus amebocyte lysate (LAL) assay revealed that L183/2 (1.3 x104 EU/mL) and Mb25 (8.9 x 103 EU/mL) had significantly higher toxicity than Epp63 (3.8 x 103 EU/mL; p<0.0001 when compared to L183/2 and Mb25) or Epp63 mutant OS strains (p<0.0001 when compared to L183/2 and Mb25). LOS truncation (from mutant Epp63 strains) affected bacterial interaction with mammalian cells, with increasing truncation inversely correlating with bacterial adherence to Chang conjunctival and HeLa cells. Adherence assays revealed that the Epp63 strain had lower adherence levels than Mb25 or L183/2 strains to Chang or HeLa cells, which decreased with OS truncation. L183/2 was found to exhibit higher levels of adherence than Mb25 or Epp63 to the cell lines. These observations collectively indicate that the OS moiety of the LOS is a significant component in facilitating membrane integrity to preserve normal cell growth and colonisation, maintaining complement resistance of the bacteria to bovine serum and susceptibility towards hydrophobic agents/antibiotics. Interestingly, it also suggests that the OS may possibly play a role in toxicity, at least as measured by LAL assay (lipid A structure of the mutants needs to be elucidated in-order to conclusively indicate OS role). Unlike M. bovis, there is already substantial information known about M. catarrhalis LOS. This information has been exploited here towards vaccine studies utilising M. catarrhalis LOS. Firstly, we investigated the biological role of M. catarrhalis LOS to confirm its suitability as a vaccine antigen. The outer membrane glycan from M. catarrhalis 2951 and 3292 wild-type and 2951lgt1/4Δ mutant strains was isolated, O-deacylated and conjugated to an outer membrane protein (rOMP26VTAL) from NTHi via an adipic dihydrazide linker; to produce the vaccine candidates referred to as 2951dLOS-rOMP26VTAL, 3292dLOS-rOMP26VTAL and 2951lgt1/4ΔdLOS-rOMP26VTAL, respectively. This allowed us to design a single vaccine that would be effective against both M. catarrhalis and H. influenzae. Three subcutaneous immunizations using 2951dLOS-rOMP26VTAL and 3292dLOS-rOMP26VTAL induced an antibody response to their respective serotype antigens and to rOMP26VTAL and NTHi. Confirming the efficacy of the conjugate vaccine, three subcutaneous immunizations using 2951lgt1/4ΔdLOS-rOMP26VTAL elicited an antigen-specific IgG response to all antigens tested (antigens from serotypes A, B, rOMP26VTAL and NTHi). Antisera from serotype A and B conjugates induced bactericidal activity against their respective serotype-specific strains, with no cross-reactivity observed. Antisera from 2951lgt1/4ΔdLOS-rOMP26VTAL immunisation showed protective efficacy against serotype A and B strains. These results indicate that 2951lgt1/4ΔdLOS-rOMP26VTAL is a promising vaccine candidate, but requires further investigation in a challenge model. Overall, the studies within this thesis show that M. bovis OS has a role in bacterial attachment, sensitivity towards bactericidal activity of bovine serum and endotoxin activity of tested strains. This thesis also shows that M. catarrhalis LOS is an important bacterial antigen that can be utilised for vaccine development. Additionally, OMP26, being highly-conserved across NTHi strains and immunogenic makes it the ideal carrier protein for respiratory related vaccine development. Combining the LOS from M. catarrhalis and the rOMP26VTAL from NTHi paves the path for the development of a single vaccine that may prevent respiratory infections caused by these two predominant pathogens.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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L’ensemble des cellules, qu’elles soient procaryotes ou eucaryotes, est doté d’une couche de glycosylation externe riche et diversifiée, composant la face dominante immédiate en relation à leur environnement. Elles résultent de processus enzymatiques complexes liant les sucres entre eux et sur des protéines ou lipides. Des variations du « glycome » peuvent apparaître dans certaines pathologies. Les cancers sont les pathologies les plus fréquentes présentant des anomalies de ces glycosylations. Ces altérations sont quasi systématiques à la surface des cellules cancéreuses. Parmi celles-ci, l’antigène Thomsen-nouveau (Tn), un N-acétylgalactosamine (GalNAc) sur une sérine ou une thréonine, est fortement exprimé dans 90% des carcinomes mammaires ainsi que dans les cancers de la vessie, du col de l’utérus, de l’ovaire, du colon, de l’estomac et de la prostate. L’omniprésence de l’antigène Tn dans de nombreux cancers, associés à son absence dans les cellules saines, en fait une cible de choix pour la thérapie ciblée ou des vaccins synthétiques antitumoraux. Aucun anticorps ciblant l’antigène Tn n’est à ce jour disponible du fait de la difficulté à développer un anticorps avec une telle spécificité. Ainsi, nous nous sommes intéressés à une autre stratégie de ciblage, basée sur l’utilisation d’une molécule capable de reconnaître l’antigène Tn. Les lectines de type C sont une famille de protéines capables de se lier spécifiquement et de façon réversible à certains glucides, en présence de calcium. La macrophage galactose lectine (MGL) est une lectine de type C ayant une affinité très importante pour le GalNac et ses dérivés comme l’antigène Tn. Ce travail a consisté, dans un premier temps, à l’utilisation d’une forme recombinante soluble de la MGL pour valider le potentiel de cet outil pour le ciblage des cellules cancéreuses. Les différentes expériences, in vitro et in vivo, impliquant la MGL, ont démontré la capacité de cette dernière à cibler spécifiquement les tumeurs humaines via l’antigène Tn. La partie extracellulaire de la MGL est de ce fait un très bon candidat de vecteur pour le diagnostic et l’imagerie de tumeurs humaines et potentiellement pour l’administration de médicaments. Dans un deuxième temps, diverses stratégies de développement d’un outil bifonctionnel exploitant cette lectine ont été exploré. Le but était de créer une plateforme peptidique fonctionnalisable d’une part avec plusieurs domaines lectines, afin de contrôler l’affinité de reconnaissance, et d’autre part des groupements fonctionnels variable selon l’application recherché (diagnostique, thérapeutique, ...). Les différentes stratégies de couplage employées nous ont permis d’accrocher plusieurs CRD de lectine sur un support peptidique, cela en conservant l’état tridimensionnel et fonctionnel des protéines. Les caractérisations effectuées démontrent une importante augmentation de l’affinité directement fonction du nombre de lectine ajouté sur la plateforme. Ce travail ouvre la voie vers de nouveaux systèmes de ciblage des sucres modulable à façon
All cells, whether prokaryotic or eukaryotic, have a rich and diversified external glycosylation layer, forming the immediate dominant face in relation to their environment. They result from complex enzymatic processes linking sugars to each other and to proteins or lipids. Variations of the "glycome" can appear in certain pathologies. Cancers are the most frequent pathologies with abnormalities in these glycosylations. These alterations are almost systematic on the surface of cancer cells. Among them, the Thomsen-new antigen (Tn), an N-acetylgalactosamine (GalNAc) on a serine or threonine, is strongly expressed in 90% of mammary carcinomas as well as in cancers of the bladder, cervix, ovary, colon, stomach and prostate. The ubiquitous presence of the Tn antigen in many cancers, combined with its absence in healthy cells, makes it a target of choice for targeted therapy or synthetic anti-tumor vaccines. No antibody targeting the Tn antigen is currently available because of the difficulty in developing an antibody with such specificity. Thus, we were interested in an alternative targeting strategy, based on the use of a molecule capable of recognizing the Tn antigen. C-Type lectins are a family of proteins capable of specifically and reversibly binding to certain carbohydrates in the presence of calcium. Macrophage galactose lectin (MGL) is a C-type lectin with a high affinity for GalNac and its derivatives such as the Tn antigen. This work consisted, initially, in the use of a soluble recombinant form of MGL to validate the potential of this tool for the targeting of cancer cells. The different experiments, in vitro and in vivo, involving MGL, demonstrated the latter's ability to specifically target human tumors via the Tn antigen. The extracellular portion of MGL is therefore a very good vector candidate for the diagnosis and imaging of human tumors and potentially for drug delivery. In a second step, various strategies for the development of a bifunctional tool exploiting this lectin were explored. The goal was to create a peptide platform that could be functionalized on one hand with several lectin domains, in order to control recognition affinity, and on the other hand with functional groups that could be variable according to the application (diagnostic, therapeutic, ...). The different coupling strategies employed allowed us to attach several lectin CRDs to a peptide support, while preserving the three-dimensional and functional state of the proteins. The characterizations carried out show a significant increase in affinity directly related to the number of lectins added to the platform. This work paves the way to new customizable sugar-targeting systems

Campylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry. Herein we describe the existence and heterogeneity of multiple LOS forms in C. jejuni strains of human and chicken origin grown at 37°C and 42°C. The C. jejuni NCTC 11168 original isolate (11168-O) was compared to the genome-sequenced variant (11168-GS), and both were found to have a lower-Mr LOS form, which was different in size and structure to the previously characterized higher-Mr form bearing GM1 mimicry. The lower-Mr form production was found to be dependent on the growth temperature as the production of this form increased from ~5 %, observed at 37°C to ~35 % at 42°C. The structure of the lower-Mr form contained a Galβ1,3GalNAc disaccharide moiety which is consistent with the termini of the GM1, asialo-GM1, GD1, GT1 and GQ1 gangliosides, however, it did not display GM1 mimicry as assessed in blotting studies but was shown by NMR analysis to resemble asialo-GM1. The production of multiple LOS forms and lack of GM1 mimicry was not a result of phase variation in the genes tested for NCTC 11168 and was also observed in most of the human and chicken isolates of C. jejuni tested.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Mycobacterium marinum est une mycobactérie pathogène des ectothermes génétiquement proche de M. tuberculosis. Ce modèle peut être utilisé pour mieux comprendre la formation des granulomes tuberculeux et déterminer le rôle des glycoconjugués pariétaux dans la régulation de la réponse immunitaire. Dans ce contexte, nos travaux ont porté sur la purification des glycolipides polaires et apolaires extraits de la paroi de M. marinum et leur caractérisation en utilisant la résonance magnétique nucléaire et la spectrométrie de masse. Nos résultats concernant l’analyse structurale de la famille des lipooligosaccharides polaires (LOSs, comprenant les LOS-I à LOS-IV) ont démontré la présence de monosaccharides rares, dont le caryophyllose et un monosaccharide N-acylé, spécifique du LOS-IV. Nous avons également observé que tous les LOSs inhibent la sécrétion macrophagique d’une cytokine pro-inflammatoire, le TNF-α. En revanche, seul le LOS-IV stimule l’expression d’antigènes (ICAM-1 et CD40) à la surface des macrophages et la production de chimiokine (IL-8). Cet effet inducteur du LOS-IV pourrait être lié à la présence du monosaccharide terminal N-acylé. Par ailleurs, les autres glycolipides polaires analysés, incluant les phosphatidyl-myo-inositol mannosides (PIM), le lipomannane (LM) et le lipoarabinomannane (LAM), possèdent des structures similaires à celles de M. tuberculosis.L’étude des glycolipides apolaires a permis de préciser la structure des glycolipides phénoliques (PGL) et des tréhaloses di-mycolates (TDM). De plus, une famille de glycolipides méconnue comprenant le Di-Mycolyl-Di-Arabinoglycérol (DMDA) a été identifiée. Le DMDA présente une structure très proche de la partie terminale du mycolyl-arabinogalactane-peptidoglycanne (mAGp), macro-complexe pariétal majoritaire des mycobactéries. Des études complémentaires effectuées chez M. bovis BCG ont démontré que ce glycolipide était lié au métabolisme du mAGp, ce qui ouvre de nouvelles pistes pour la compréhension du mode d’action de certaines drogues anti-tuberculeuses telles que la thiacétazone
Mycobacterium marinum is a natural pathogen of ectotherms genetically close to M. tuberculosis. This pathogen model is useful for deciphering the role of mycobacterial cell wall glycolipids in granulomatous infection. In this context, our work focused on the purification of both polar and apolar glycolipids extracted from M. marinum cell wall and their structural characterization using nuclear magnetic resonance and mass spectrometry. Analysis of the polar lipooligosaccharide family (LOSs, including LOS-I to LOS-IV) demonstrated the presence of several rare or even unique monosaccharides including caryophyllose, derivatives and a N-acylated monosaccharide specific of LOS-IV. Biological activity assays showed that LOSs exert an important pro-inflammatory effect by decreasing the TNF-α secretion from macrophages. Moreover, LOS-IV was found to stimulate the expression of the chemokine IL-8 and cell surface antigens (CD40 and ICAM-1) on macrophages. This specific immunostimulatory property was related to the presence of the terminal N-acylated monosaccharide in LOS-IV. In addition, other polar glycolipids analyzed, including phosphatidyl-myo-inositol mannosides (PIM), lipomannane (LM) and lipoarabinomannan (LAM), possess similar structures than M. tuberculosis. The study of apolar glycolipids permitted to precise the structure of phenolic glycolipids (PGL) and trehalose di-mycolates (TDM). Moreover, a family of unusual glycolipids, including Di-Mycolyl-Di-Arabinoglycérol (DMDA), was identified. DMDA structure is very close from the terminal part of peptidoglycan-arabinogalactan-mycolyl (mAGp), the mycobacterial cell wall macro-complex. Additional studies performed in M. bovis BCG showed that this glycolipid is related to mAGp, providing new insights about the mode of action of anti-tuberculous drugs such as thiacetazone

Haemophilus parainfluenzae (Hp) and H. influenzae (Hi) are closely related members of the Pasteurellaceae family and are common commensal bacteria of the human nasopharynx. Whilst Hi is frequently implicated in meningitis, otitis media and respiratory tract infections, reports of pathogenic behaviour by Hp are very rare. Lipopolysaccharide (LPS) is a key component of the Gram negative cell wall, and its structure influences the ability of Haemophilus to interact with the host and evade immune clearance. A better understanding of the differences in LPS structure between Hi and Hp could help to ascertain which parts of the molecule are important for commensal and pathogenic behaviour. Hi LPS comprises lipid A, a conserved oligosaccharide inner core, and an oligosaccharide outer core that differs between strains. The latter is partly phase variable by the slipped strand mispairing during replication of DNA repeat tracts within several LPS biosynthesis genes. Very little was known about LPS in Hp so we investigated its biosynthesis and structure in a panel of 20 Hp carriage isolates. Using PCR, DNA sequencing and Southern analysis we demonstrated that Hp possesses homologues of the Hi lipid A and inner core LPS synthesis genes and a few of the genes for outer core synthesis; however, homologues of the Hi phase variable outer core genes were largely absent and did not contain repeat tracts. The results of immunoblotting and collaborative structural analysis were consistent with this data. Phosphocholine, a phase variable Hi LPS epitope that has been implicated in otitis media, was found to be absent in Hp LPS due to the lack of four genes required for its biosynthesis and incorporation. The introduction of these genes into Hp led to the phase variable addition of phosphocholine to the LPS, indicating that there is no fundamental reason why Hp could not use a similar mechanism of variation to Hi if it was advantageous to do so. SDS-PAGE data suggested the presence of O-antigens (repeated chains of sugars) in many of the Hp strains, an unusual feature for Haemophilus, and all of the strains were found to contain a potential O-antigen synthesis locus. Each locus encodes homologues of several glycosyltransferases in addition to either the Wzy polymerase- or ABC transporter-dependent mechanisms of O-antigen synthesis and transport. Comparisons of wild type and isogenic mutant strains showed that the O-antigen enhances resistance to complement-mediated killing and appears to affect adhesion to epithelial cells in vitro. Hp is a successful commensal organism but lacks the flexibility of adapting its LPS using repeat-mediated phase variation, potentially limiting its range of host niches.

Haemophilus parasuis est un pathogène porcin causant la maladie de Glässer caractérisée par de la polysérosite fibrineuse, polyarthrite, méningite et septicémie. La pathogenèse de l’infection et les facteurs de virulence sont encore mal connus. Le site de colonisation de Haemophilus parasuis dans le tractus respiratoire supérieur est controversé. Pour accéder à la circulation sanguine, H. parasuis doit envahir la muqueuse. H. parasuis adhère à des cellules épithéliales porcines de trachée (NPTr). Pour accéder au système nerveux central et causer la méningite, H. parasuis doit traverser la barrière hémato-méningée. H. parasuis adhère à et envahit des cellules endothéliales porcines de microvaisseaux cérébraux (PBMEC) provenant de la BBB. Le but de cette étude était d’étudier certaines interactions entre H. parasuis et son lipooligosccharide (LOS), et des cellules endothéliales et épithéliales porcines. Les résultats démontrent que l’adhésion de H. parasuis Nagasaki aux NPTr et aux PBMEC est en partie médiée par son LOS. H. parasuis induit l’apoptose des NPTr et des PBMEC, mais le LOS ne semble pas impliqué. H. parasuis, et à un niveau moindre son LOS, stimulent la sécrétion d’interleukine- (IL) 6 et d’IL-8. Différentes souches de H. parasuis sérotypes 4 et 5 (sérotypes les plus prévalents en Amérique du Nord) stimulent également les NPTr et PBMEC à produire IL-6 et IL-8. Les résultats suggèrent que le LOS de H. parasuis joue un certain rôle dans la pathogenèse de l’infection, mais d’autres composantes bactériennes sont également impliquées.
Haemophilus parasuis is a swine pathogen that causes Glässer’s disease characterized by fibrinous polyserositis, polyarthritis, meningitis and septicemia. The pathogenesis of the infection and virulence factors are not well known. Whether the upper respiratory tract is the site of colonization of H. parasuis is still a controversial issue. H. parasuis must invade the mucosa to gain access to the bloodstream. H. parasuis is able to adhere to newborn pig trachea cells (NPTr). H. parasuis must then cross the blood-brain barrier to gain access to the central nervous system in cases of meningitis. H. parasuis is able to adhere to and invade porcine brain microvascular endothelial cells (PBMEC). The aim of this work was to study the interactions between H. parasuis, its lipooligosccharide (LOS), and porcine endothelial and epithelial cells. Results showed that adhesion of H. parasuis Nagasaki to NPTr and PBMEC was partially mediated by its LOS. H. parasuis induced NPTr and PBMEC apoptosis, although purified LOS does not seem to be involved. H. parasuis, and to a lesser extent its LOS, stimulated the release of interleukin- (IL) 6 and IL-8. Field strains of H. parasuis serotypes 4 and 5 (the most prevalent serotypes in North America) also induced the production of IL-6 and IL-8. Results suggest that H. parasuis LOS plays a role in the pathogenesis of the infection, but other bacterial components are also involved.

博士
國立臺灣大學
微生物學研究所
104
Resistance to phagocyte killing is an important virulence factor in mycobacteria. Dictyostelium has been used to study the interaction between phagocytes and bacteria, given its similarity to the mammalian macrophage. Here, we investigated the genes responsible for virulence to Dictyostelium by screening 1728 transposon mutants of the Mycobacterium marinum NTUH-M6094 strain. A total of 30 mutants that were permissive for Dictyostelium growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) were located in the lipooligosaccharide (LOS) synthesis cluster. LOS are antigenic glycolipids and the core LOS structure from LOS-I to LOS-IV have been reported to exist in M. marinum. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of mmar_2318 or mmar_2319 resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of mmar_2318 or mmar_2319 confirmed that both genes contributed to virulence towards Dictyostelium but not entry and replication inside Dictyostelium. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that mmar_2318 or mmar_2319 deletion mutant could grow in macrophages, and their initial entry rate was not affected in J774a.1 but significantly increased in THP-1. In conclusion, although mmar_2319 has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, mmar_2318 that is also involved in the biosynthesis of LOS. Deletion of mmar_2318 or mmar_2319 both exhibits reduction of virulence towards Dictyostelium and increased entry into THP-1 cells.

Chlamydia trachomatis, a pathogen responsible for major diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to molecular genetic manipulation. The development of a system(s) for genetic analysis would significantly accelerate our ability to identify genes that enable Chlamydia to establish infection, survive within its host, and cause disease. This thesis describes two methods used to assess gene function in Chlamydia and to provide insights into its biology and pathogenesis. The first method described is based on specific inhibitors and is used to probe the role of lipooligosaccharide (LOS), a main lipid components of bacterial outer membranes. Using this approach, we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, blocks the synthesis of LOS in C. trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of anti-chlamydial agents.

The second part of this thesis describes the development of a system with which to perform forward genetics in C. trachomatis. Forward genetics approaches set out to identify the gene or set of genes that contributes to a specific biological process and usually entails generating random mutations in a large number of organisms, isolating mutants with an aberrant phenotype, and identifying the alleles associated with the mutant phenotype. In this approach, chemical mutagenesis is coupled with whole genome sequencing (WGS) and a system for DNA exchange within infected cells to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. We identified mutants with altered glycogen metabolism, including an attenuated strain defective for Type II secretion. The coupling of chemically induced gene variations and WGS to establish genotype-phenotype associations should be broadly applicable to the growing list of microorganisms intractable to traditional genetic mutational analysis.

Dissertation

Thesis (Ph.D.)--State University of New York at Buffalo, 2006.
Title from PDF title page (viewed on Oct. 09, 2006) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Campagnari, Anthony A. Includes bibliographical references.